Flux Capacitor and Flux Simulator

Category Cross-Omics>Next Generation Sequence Analysis/Tools, Cross-Omics>Agent-Based Modeling/Simulation/Tools and Genomics>Gene Expression Analysis/Profiling/Tools

Abstract The Flux Capacitor is a high-throughput computational tool to predict the abundances of splice-forms and Alternative Splicing (AS) events based on a reference annotation and reads from an RNA-seq experiment.

The program formulates linear constraints from both pieces of information, deconvolution and segregation into single transcript species is achieved by solving these constraints by a so-called linear program solver.

The Flux Capacitor is implemented in system independent Java technology, but requires platform-specific compilations of the linear program solver libraries.

It focuses on abundancy prediction for splice forms, transcripts and alternative splicing events from reads generated by applying new sequencing technologies (e.g., the Illumina Genome Analyzer, Roche 454-sequencing, etc.) to RNA molecules extracted from a population of cells.

RNA-Seq --

RNA-Seq also called “Whole Transcriptome Shotgun Sequencing” and dubbed “a revolutionary tool for transcriptomics”, refers to the use of High-throughput sequencing technologies to sequence cDNA in order to get information about a sample’s RNA content, a technique that is quickly becoming invaluable in the study of diseases like cancer.

Thanks to the deep coverage and base level resolution provided by next-generation sequencing instruments, RNA-Seq provides researchers with efficient ways to measure transcriptome data experimentally, allowing them to get information such as, how different alleles of a gene are expressed, detect post-transcriptional mutations or identifying gene fusions.

A comprehensive in silico simulation of all steps in an RNA-Seq experiment is provided by the Flux Simulator (see below...).

The Flux Capacitor --

The input for the Flux Capacitor is the annotation of a reference transcriptome and reads from RNA-seq technologies aligned to the genome.

From the reference annotation, “splicing graphs” are produced and reads are mapped to corresponding edges in these graphs according to the position where they align in the genomic sequence.

The resulting graph with edges labeled by the number of reads can be interpreted as a ‘flow network’ where each transcript representing a transportation path from its start to its end and consequently each edge a possibly shared segment of transportation along which a certain number of reads per nucleotide -- i.e., a flux -- is observed.

Given a density function of reads along a transcript, the expected participation of each transcript in an edge under consideration can be estimated.

The basic idea is to cast back from these latter participations and the observed number of reads - allowing for a certain amount of noise - to the original transcript abundances.

To do so, a linear constraint is formalized for each edge, and an optimal solution for the complete set of constraints is found by a standard linear program solver.

The Flux Simulator --

The Flux Simulator is the part of the FLUX project that aims at providing an in silico reproduction of the experimental pipelines for RNA-Seq, adopting a minimal set of parameters.

Corresponding models were established after analyzing RNA-Seq experiments from different cell types, sample preparation protocols and sequencing platforms.

The first step of the FLUX project is in fact a transcriptome simulator. Subsequently, common sources of systematic bias in the abundance and distribution of produced reads are mimicked-whether they incur during library construction, or, in the sequencing process.

The Flux Simulator provides a flexible base to design ‘benchmark’ experiments based on the new sequencing technologies, as for instance, abundance predictions of the Flux Capacitor.

The Flux Simulator is an in silico simulation (as stated above...) of the biological and experimental background that influences the distribution of reads in an RNA-seq experiment.

Starting by assigning a generic ‘gene expression’ profile for the interrogated cells, the Flux Simulator can mimic different orders and parameters of standard sequencing preparation protocols.

In the end, read-distributions are presented visually and the obtained reads can be used to evaluate abundancy prediction methods.

Simulating RNA-seq experiments --

The simulation process will Not give you an identical reproduction of the processes that happen in corresponding experiments.

Hence, do Not assume that the output of this program can substitute real data for purposes of training.

The Flux Simulator gives an impression of differential systematic biases that can be introduced in biological experiments.

It has been designed as an in silico benchmarking environment that - as close as current knowledge permits - mimics scenarios that happen in RNA-seq experiments.

It can also be seen as a tool to understand laboratory techniques -- as such, often lack data from intermediate steps -- and hopefully helps to improve these.

The Flux Simulator separates each RNA-seq experiment -- a so-called “run” -- into three (3) different steps, i.e., gene expression, library construction, and sequencing.

Together with the initial assumption of a reference annotation, i.e., transcript structures, the total process comprises four (4) steps which combined, are representing the consecutive pipeline of a run, described by a specific parameter file.

The status of each step is indicated by a color dot, grey means the module is missing data or obligatory parameters, red means input data/parameters are ready, but the simulated experiment has Not yet been performed, and green means the corresponding step has been finished.

“Run” and “Stop” buttons refer to the currently selected tab and initiate or end the corresponding simulation step.

A typical starting point is to either load and copy data of an existing Run, or, to start creating a new project specifying location of the annotation and project files.

Finally, at the bottom of the process window there is a status bar which indicates the progress while simulations are carried out.

Flux Capacitor and Flux Simulator documentation --

Extensive documentation is supplied to the user via a Wiki for the Flux Capacitor and Flux Simulator.

System Requirements

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Manufacturer Web Site Flux Capacitor and Flux Simulator

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G6G Abstract Number 20774

G6G Manufacturer Number 104298